Handbook of Microbiological Media, Fourth Edition part 95 potx

Preparation of Medium: Add components, except IPTG solution, to distilled/deionized water and bring volume to 990.0mL.. 0.01g Preparation of Antibiotic Solution: Add components to distil

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LB Medium with Tetracycline and Ampicillin 935

Preparation of Medium: Add components to distilled/deionized

water and bring volume to 1.0L Mix thoroughly Adjust pH to 7.0

Dis-tribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–

121°C

Use: For the cultivation and maintenance of Escherichia coli.

LB Medium with IPTG Medium

Compositionper liter:

NaCl 10.0g

Pancreatic digest of casein 10.0g

Yeast extract 5.0g

IPTG solution 10.0mL

pH 7.0 ± 0.2 at 25°C

IPTG Solution:

Compositionper 10.0mL:

IPTG (Isopropylthio-β-galactoside) 0.24g

Preparation of IPTG Solution: Add IPTG to distilled/deionized

water and bring volume to 10.0mL Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except IPTG solution, to

distilled/deionized water and bring volume to 990.0mL Mix thoroughly

Adjust pH to 7.0 Autoclave for 15 min at 15 psi pressure–121°C

Asep-tically add sterile IPTG solution Mix thoroughly AsepAsep-tically distribute

into sterile tubes or flasks

LB Medium with Kanamycin

NaCl 10.0g

Yeast extract 5.0g

Kanamycin solution 50.0mL

Kanamycin Solution:

Kanamycin 50.0mg

Preparation of Kanamycin Solution : Add kanamycin to

dis-tilled/deionized water and bring volume to 50.0mL Mix thoroughly

Filter sterilize Warm to 50°C

Preparation of Medium: Add components, except kanamycin

so-lution, to distilled/deionized water and bring volume to 950.0mL Mix

thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to

50–55°C Aseptically add 50.0mL of sterile kanamycin solution Mix

thoroughly Aseptically distribute into sterile tubes or flasks

Use: For the cultivation of Escherichia coli.

LB Medium with 25mg of Kanamycin

(ATCC Medium 1236)

NaCl 10.0g

Yeast extract 5.0g

Kanamycin 0.025g

pH 7.0 ± 0.2 at 25°C

wa-ter and bring volume to 1.0L Mix thoroughly Adjust pH to 7.0 Distribute

into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C

NaCl 10.0g Pancreatic digest of casein 10.0g Yeast extract 5.0g Kanamycin 0.05g

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 7.0 Dis-tribute into tubes or flasks Autoclave for 15 min at 15 psi pressure– 121°C

NaCl 10.0g Pancreatic digest of casein 10.0g Yeast extract 5.0g Kanamycin 0.1g

pH 7.0 ± 0.2 at 25°C

Use: For the cultivation and maintenace of Erwinia uredovora.

LB Medium with Rifampicin

NaCl 10.0g Pancreatic digest of casein 10.0g Yeast extract 5.0g Rifampicin 0.1g

pH 7.0 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Enterobacter cloacae.

LB Medium with Tetracycline

NaCl 10.0g Pancreatic digest of casein 10.0g Yeast extract 5.0g Tetracycline 0.02g

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized wa-ter and bring volume to 1.0L Mix thoroughly Adjust pH to 7.0 Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C

LB Medium with Tetracycline and Ampicillin

NaCl 10.0g Pancreatic digest of casein 10.0g

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936 LB Medium with Tetracycline and Ampicillin

Yeast extract 5.0g

Antibiotic solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Antibiotic Solution:

Ampicillin 0.01g

Tetracycline 0.01g

Preparation of Antibiotic Solution: Add components to distilled/

deionized water and bring volume to 10.0mL Mix thoroughly Filter

sterilize

Preparation of Medium: Add components, except antibiotic

solu-tion, to distilled/deionized water and bring volume to 990.0mL Mix

thoroughly Adjust pH to 7.0 Autoclave for 15 min at 15 psi pressure–

121°C Cool to 45°–50°C Aseptically add sterile antibiotic solution

Mix thoroughly Aseptically distribute into sterile tubes or flasks

LB Medium with Tetracycline and Ampicillin

NaCl 10.0g

Yeast extract 5.0g

pH 7.0 ± 0.2 at 25°C

Ampicillin 0.01g

Tetracycline 5.0mg

sterilize

thoroughly Adjust pH to 7.0 Autoclave for 15 min at 15 psi pressure–

121°C Cool to 45°–50°C Aseptically add sterile antibiotic solution

Mix thoroughly Aseptically distribute into sterile tubes or flasks

LB Medium with Thiamine Monophosphate

See: LB Medium with TMP

LB Medium with Thiamine Pyrophosphate

See: LB Medium with TPP

LB Medium with TMP (LB Medium with Thiamine Monophosphate)

NaCl 10.0g

Yeast extract 5.0g

Thiamine monophosphate 0.038mg

pH 7.0 ± 0.2 at 25°C

Dis-tribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–

121°C

LB Medium with TPP (LB Medium with Thiamine Pyrophosphate)

NaCl 10.0g Pancreatic digest of casein 10.0g Yeast extract 5.0g Thiamine pyrophosphate 0.046mg

pH 7.0 ± 0.2 at 25°C

NaCl 10.0g Pancreatic digest of casein 10.0g Yeast extract 5.0g Glucose solution 10.0mL Diaminopimelic acid solution 10.0mL Thymidine solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Glucose Solution:

D-Glucose 0.8g

Preparation of Glucose Solution: Add D-glucose to distilled/de-ionized water and bring volume to 10.0mL Mix thoroughly Filter ster-ilize

Diaminopimelic Acid Solution:

DL-Diaminopimelic acid 0.1g

Preparation of Diaminopimelic Acid Solution: Add diamin-opimelic acid to distilled/deionized water and bring volume to 10.0mL Mix thoroughly Filter sterilize

Thymidine Solution:

Thymidine 0.02g

Preparation of Thymidine Solution: Add thymidine to distilled/ deionized water and bring volume to 10.0mL Mix thoroughly Filter sterilize

Preparation of Medium: Add components—except glucose solu-tion, diaminopimelic acid solusolu-tion, and thymidine solution—to dis-tilled/deionized water and bring volume to 970.0mL Mix thoroughly Gently heat and bring to boiling Autoclave for 15 min at 15 psi pres-sure–121°C Cool to 45°–50°C Aseptically add sterile glucose solu-tion, diaminopimelic acid solusolu-tion, and thymidine solution Mix thoroughly Aseptically distribute into sterile tubes or flasks

Use: For the cultivation and maintenance of Bacillus subtilis and Escherichia coli.

and Ampicillin

NaCl 10.0g Pancreatic digest of casein 10.0g Yeast extract 5.0g

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LB Top Agar 937

Glucose solution 10.0mL

Diaminopimelic acid solution 10.0mL

Thymidine solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Ampicillin 0.01g

Tetracycline 0.01g

sterilize

D-Glucose 0.8g

Preparation of Glucose Solution: Add D-glucose to

distilled/de-ionized water and bring volume to 10.0mL Mix thoroughly Filter

ster-ilize

Diaminopimelic Acid Solution:

DL-Diaminopimelic acid 0.1g

Preparation of Diaminopimelic Acid Solution: Add

diamin-opimelic acid to distilled/deionized water and bring volume to 10.0mL

Mix thoroughly Filter sterilize

Thymidine Solution:

Thymidine 0.02g

Preparation of Thymidine Solution: Add thymidine to distilled/

sterilize

Preparation of Medium: Add components—except glucose

solu-tion, diaminopimelic acid solusolu-tion, and thymidine solution—to

dis-tilled/deionized water and bring volume to 960.0mL Mix thoroughly

Gently heat and bring to boiling Autoclave for 15 min at 15 psi

pres-sure–121°C Cool to 45°–50°C Aseptically add sterile antibiotic

solu-tion, glucose solusolu-tion, diaminopimelic acid solusolu-tion, and thymidine

solution Mix thoroughly Aseptically distribute into sterile tubes or

flasks

Use: For the cultivation and maintenance of Bacillus subtilis and

Escherichia coli.

LB Modified Broth (ATCC Medium 1620)

Composition per 1030.4mL:

Tryptone 10.0g

NaCl 5.8g

Yeast extract 5.0g

NaCl Solution:

NaCl 20.0g

Preparation of NaCl Solution: Add NaCl to distilled/deionized

water and bring volume to 100.0mL Mix thoroughly Autoclave for 15

min at 15 psi pressure–121°C

MgCl 2 Solution:

MgCl2 0.9g

Preparation of MgCl 2 Solution: Add MgCl2 to distilled/deion-ized water and bring volume to 10.0mL Mix thoroughly Autoclave for

15 min at 15 psi pressure–121°C

CaCl2 Solution:

CaCl2 0.5g

Preparation of CaCl2 Solution: Add CaCl2 to distilled/deionized water and bring volume to 10.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C

D-Glucose 40.0g

Preparation of Glucose Solution: Add glucose to distilled/deion-ized water and bring volume to 100.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C

Preparation of Medium: Add components except salts and glucose solutions to distilled/deionized water and bring volume to 1.0L Mix thoroughly Autoclave for 30 min at 15 psi pressure–121°C Cool to 45°–50°C Aseptically add 16.8mL NaCl solution, 1.6 mL MgCl2 so-lution, 2.0mL CaCl2 solution, and 10.0mL glucose solution Mix thor-oughly

Use: For the cultivation of Escherichia coli (Migula) Castellani and

Chalmers

LB Streptomycin Medium

NaCl 10.0g Pancreatic digest of casein 10.0g Yeast extract 5.0g Streptomycin solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Streptomycin Solution:

Streptomycin 0.2g

Preparation of Streptomycin Solution: Add streptomycin to dis-tilled/deionized water and bring volume to 10.0mL Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except streptomycin solution, to distilled/deionized water and bring volume to 990.0mL Mix thoroughly Adjust pH to 7.0 Autoclave for 15 min at 15 psi pres-sure–121°C Cool to 45°–50°C Aseptically add sterile streptomycin solution Mix thoroughly Aseptically distribute into sterile tubes or flasks

LB Top Agar

Pancreatic digest of casein 10.0g Agar 7.0g NaCl 5.0g Yeast extract 5.0g

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Gently heat and bring

to boiling Adjust pH to 7.0 Autoclave for 15 min at 15 psi pressure– 121°C Cool to 50°C Distribute into flasks in 100.0mL volumes Reautoclave for 15 min at 15 psi pressure–121°C Store at 25°C

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938 LBE Medium

Use: For use as a top agar for the distribution of bacteriophage or

Escherichia coli.

LBE Medium

NaCl 10.0g

Yeast extract 5.0g

Glucose solution 10.0mL

50X medium E 4.0mL

pH 7.0 ± 0.2 at 25°C

D-Glucose 20.0g

Preparation of Glucose Solution: Add D-glucose to

distilled/de-ionized water and bring volume to 100.0mL Mix thoroughly Filter

sterilize

50X Medium E:

K2HPO4, anhydrous 500.0g

Na(NH4)HPO4·4H2O 175.0g

Citric acid·H2O 100.0g

MgSO4·7H2O 10.0g

Preparation of 50X Medium E: Add components to 670.0mL of

distilled/deionized water in the following order: MgSO4·7H2O, citric

acid·H2O, K2HPO4, and Na(NH4)HPO4·4H2O Mix thoroughly Bring

volume to 1.0L with distilled/deionized water

Preparation of Medium: Add components—except glucose

solu-tion and 50X medium E—to distilled/deionized water and bring

vol-ume to 986.0mL Mix thoroughly Autoclave for 15 min at 15 psi

pressure–121°C Cool to 45°–50°C Aseptically add 10.0mL of sterile

glucose solution and 4.0mL of sterile 50X medium E Mix thoroughly

Aseptically distribute into sterile tubes or flasks

LBS™ Agar

(Lactobacillus Selection Agar)

Composition per liter:

Sodium acetate·3H2O 25.0g

Glucose 20.0g

Agar 15.0g

KH2PO4 6.0g

Yeast extract 5.0g

Ammonium citrate 2.0g

Polysorbate 80 1.0g

MgSO4 0.575g

FeSO4 0.034g

MnSO4 0.12g

Acetic acid, glacial 1.32mL

pH 5.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD

Di-agnostic Systems

Preparation of Medium: Add components, except acetic acid, to

distilled/deionized water and bring volume to 998.7mL Mix

thorough-ly Gently heat and bring to boiling Add glacial acetic acid Mix

thor-oughly Gently heat while stirring and bring to 90°–100°C for 2–3 min

Do not autoclave Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the selective isolation, cultivation, and enumeration of lacto-bacilli

LBS™ Broth

(Lactobacillus Selection Broth)

Sodium acetate·3H2O 25.0g Glucose 20.0g Pancreatic digest of casein 10.0g

KH2PO4 6.0g Yeast extract 6.0g Ammonium citrate 2.0g Polysorbate 80 1.0g MgSO4 0.575g FeSO4 0.034g MnSO4 0.12g Acetic acid, glacial 1.32mL

pH 5.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Di-agnostic Systems

Preparation of Medium: Add components, except acetic acid, to distilled/deionized water and bring volume to 998.7mL Mix

thorough-ly Gently heat and bring to boiling Add glacial acetic acid Mix thor-oughly Gently heat while stirring and bring to 90°–100°C for 2–3 min

Do not autoclave Aseptically distribute into sterile tubes

Use: For the selective isolation and cultivation of lactobacilli

LBS Oxgall Agar

See: Lactobacillus Selection Oxgall Agar

LC Broth

NaCl 10.0g Pancreatic digest of casein 10.0g Yeast extract 5.0g Glucose 1.0g CaCl2·2H2O (1M solution) 5.0mL

MgSO4·7H2O (1M solution) 5.0mL

pH 7.4 ± 0.2 at 25°C

CaCl 2 ·2H 2 O Solution:

CaCl2·2H2O 14.7g

Preparation of CaCl 2 ·2H 2 O Solution: Add CaCl2·2H2O to dis-tilled/deionized water and bring volume to 100.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C

MgSO4·7H2O Solution:

MgSO4·7H2O 24.65g

Preparation of MgSO4·7H2O Solution: Add MgSO4·7H2O to distilled/deionized water and bring volume to 100.0mL Mix

thorough-ly Autoclave for 15 min at 15 psi pressure–121°C

Preparation of Medium: Add components, except CaCl2·2H2O so-lution and MgSO4·7H2O solution, to distilled/deionized water and bring volume to 990.0mL Mix thoroughly Adjust pH to 7.4 Auto-clave for 15 min at 15 psi pressure–121°C Cool to 50°C Aseptically

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Lecithin Agar 939

add 5.0mL of sterile CaCl2·2H2O solution and 5.0mL of sterile

MgSO4·7H2O solution Mix thoroughly Aseptically distibute into

ster-ile tubes or flasks

LD Agar

See: Lombard-Dowell Agar

LD Bile Agar

See: Lombard-Dowell Bile Agar

LD Broth

See: Lombard-Dowell Broth

LD Egg Yolk Agar

See: Lombard-Dowell Egg Yolk Agar

LD Esculin Agar

See: Lombard-Dowell Esculin Agar

LD Esculin HiVeg Agar (Lombard-Dowell Esculin Agar, HiVeg)

Agar 20.0g

Plant hydrolysate No 1 5.0g

Yeast extract 5.0g

NaCl 2.5g

Esculin 1.0g

Ferric citrate 0.5g

L-Cystine 0.4g

L-Tryptophan 0.2g

Fe4(P2O7)3·H2O 0.01g

Vitamin K1 0.01g

pH 7.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from

HiMe-dia

water and bring volume to 1.0L Mix thoroughly Distribute into tubes

or flasks Autoclave for 15 min at 15 psi pressure–121°C Pour into

sterile Petri plates

Use: For the cultivation of a wide variety of anaerobic bacteria For the

differentiation of anaerobic bacteria based on esculin hydrolysis, H2S

production, and catalase production Bacteria that hydrolyze esculin

appear as colonies surrounded by a red-brown to dark brown zone

Bacteria that produce H2S appear as black colonies

LD Gelatin Agar

See: Lombard-Dowell Gelatin Agar

LD HiVeg Agar (Lombard-Dowell Agar, HiVeg)

Agar 20.0g

Plant hydrolysate 5.0g

Yeast extract 5.0g

NaCl 2.5g

L-Cystine 0.4g

L-Tryptophan 0.2g

Na2SO3 0.1g

Vitamin K1 0.01g

Fe4(P2O7)3·H2O 0.01g

pH 7.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMe-dia

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Distribute into tubes

or flasks Autoclave for 15 min at 15 psi pressure–121°C Pour into sterile Petri plates

Use: For the cultivation and identification of a variety of obligate anaerobic

bacteria For the cultivation of Bacteroides species, Fusobacterium species, Clostridium species, and nonspore-forming Gram-positive anaerobes.

Lead Acetate Agar

Agar 15.0g Peptone 15.0g Proteose peptone 5.0g Glucose 1.0g Lead acetate 0.2g

Na2S2O3 0.08g

pH 6.6 ± 0.2 at 25°C

to boiling Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C Pour into sterile Petri dishes or leave in tubes Allow tubes to cool in a slanted position

Use: For the cultivation and differentiation of Gram-negative coliform bacteria based on H2S production Bacteria that produce H2S turn the medium brown

LEB, FDA

See: Listeria Enrichment Broth, FDA

Lecithin Agar

Fraction B 500.0mL Fraction A 450.0mL Fraction C 50.0mL

pH 7.2 ± 0.2 at 25°C

Fraction A:

Agar 18.0g Tryptone 10.0g Yeast extract 5.0g Glucose 5.0g

Preparation of Fraction A: Add components to distilled/deionized water and bring volume to 500.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to 43°C

Fraction B:

Crude soy lecithin 30.0g

Preparation of Fraction B: Add crude soy lethicin to distilled/de-ionized water and bring volume to 450.0mL Mix thoroughly Gently heat and bring to boiling Swirl to form a viscous sol Sonicate until ho-mogeneous Blending of unheated fraction A in a Waring blender for 2 min at high speed is also satisfactory Autoclave for 15 min at 15 psi pressure–121°C Cool to 43°C

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940 Lecithin HiVeg Agar

Fraction C:

CaCl2 0.6g

Preparation of Fraction C: Add CaCl2 to distilled/deionized water

and bring volume to 50.0mL Mix thoroughly Autoclave for 15 min at

15 psi pressure–121°C Cool to 43°C

Preparation of Medium: Combine fractions with gentle swirling

To prevent separation, immediately pour into sterile Petri plates

Use: For the detection of microbial phospholipases

Lecithin HiVeg Agar

Agar 20.5g

Papaic digest of soybean meal 5.0g

Polysorbate 80 5.0g

NaCl 5.0g

Na2S2O3 1.0g

L-Histidine 1.0g

Lecithin 0.7g

pH 7.3 ± 0.2 at 25°C

HiMe-dia

or flasks Autoclave for 15 min at 15 psi pressure–121°C Pour into

sterile Petri plates

Use: For the detection of bacterial contamination of surfaces in

unpro-tected and prounpro-tected areas

Lecithin Lactose Agar

Agar 15.0g

Lactose 10.0g

NaCl 5.5g

Peptic digest of animal tissue 5.5g

Yeast extract 3.9g

Pancreatic digest of heart muscle 3.3g

Cornstarch 1.1g

Egg lecithin 0.66g

L-Cysteine·HCl·H2O 0.5g

NaN3 0.2g

Neomycin sulfate 0.15g

CaCl2 0.05g

Bromcresol Purple 0.02g

pH 6.8 ± 0.2 at 25°C

Caution: Sodium azide is toxic Azides also react with metals and

disposal must be highly diluted

Source: This medium is available as a prepared medium from BD

Di-agnostic Systems

water and bring volume to 1.0L Mix thoroughly Gently heat and bring

to boiling Distribute into tubes or flasks Autoclave for 15 min at 15

psi pressure–121°C Pour into sterile Petri dishes

Use: For the isolation, cultivation, and differentiation of histolytic clostridia from clinical specimens based on lecithinase production and

lac-tose fermentation It is especially useful for the differentiation of Clostrid-ium perfringens, ClostridClostrid-ium sordelli, ClostridClostrid-ium novyi, ClostridClostrid-ium sep-ticum, and Clostridium histolyticum Bacteria that produce lecithinase

appear as colonies surrounded by an opalescent zone Bacteria that ferment lactose appear as colonies surrounded by a yellow zone

Lecithin Lipase Anaerobic Agar

Pancreatic digest of casein 40.0g Agar 25.0g Yeast extract 5.0g

Na2HPO4·12H2O 5.0g Glucose 2.0g NaCl 2.0g

KH2PO4 1.0g MgSO4·7H2O 0.1g Egg yolk emulsion 100.0mL

pH 7.6 ± 0.2 at 25°C

Egg Yolk Emulsion:

Composition: Chicken egg yolks 11 Whole chicken egg 1

Preparation of Egg Yolk Emulsion: Soak eggs with 1:100 dilu-tion of saturated mercuric chloride soludilu-tion for 1 min Crack eggs and separate yolks from whites Mix egg yolks with 1 chicken egg Filter sterilize

Preparation of Medium: Add components, except egg yolk emul-sion, to distilled/deionized water and bring volume to 900.0mL Mix thoroughly Gently heat and bring to boiling Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C Aseptically add sterile egg yolk emulsion Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the isolation, cultivation, and differentiation of Clostridium

species based on lecithinase production and lipase production Bacteria that produce lecithinase appear as colonies surrounded by a zone of insoluble precipitate Bacteria that produce lipase appear as colonies with a pearly iridescent sheen

Lecithin Tween™ Medium (LT Medium)

Tween™ 80 30.0g Agar 15.0g Pancreatic digest of casein 10.0g Peptic digest of animal tissue 10.0g NaCl 5.0g Lecithin 5.0g

Na2S2O3·5H2O 5.0g Glycerol 3.0g Histidine, free base 1.0g Glucose 1.0g

pH 7.5 ± 0.2 at 25°C

5–Fluorocytosine 0.2g Fosfomicin 0.1g Ticarcillin 0.1g

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Lee's Multidifferential HiVeg Agar 941

sterilize

thoroughly Gently heat and bring to boiling Autoclave for 15 min at

15 psi pressure–121°C Cool to 45°–50°C Aseptically add sterile

anti-biotic solution Mix thoroughly Pour into sterile Petri dishes in

20.0mL volumes

Use: For the isolation and cultivation of multiresistant lipophilic

Corynebacterium species, especially Corynebacterium group JK found

primarily in infections in immunocompromised hosts and patients with

prosthetic valve endocarditis

Lee’s Agar

Agar 18.0g

Yeast extract 10.0g

Lactose 5.0g

Sucrose 5.0g

CaCO3 3.0g

K2HPO4 0.5g

Bromcresol Purple (0.2% solution) 10.0mL

pH 7.0 ± 0.2 at 25°C

Bromcresol Purple Solution:

Preparation of Bromcresol Purple Solution: Add Bromcresol

Purple to distilled/deionized water and bring volume to 10.0mL Mix

thoroughly Filter sterilize

Preparation of Medium: Add components, except Bromcresol

Pur-ple solution, to distilled/deionized water and bring volume to 990.0mL

Mix thoroughly Adjust pH to 7.0 Gently heat and bring to boiling

Au-toclave for 20 min at 15 psi pressure–121°C Cool to 45°–50°C

Asepti-cally add sterile Bromcresol Purple solution Mix thoroughly Pour into

sterile, chilled Petri dishes in 20–25mL volumes Swirl flask while

dis-pensing to evenly suspend CaCO3 Dry plates at 30°C for 18–24 hr

be-fore use

Use: For the isolation, cultivation, and enumeration of Lactobacillus

bulgaricus from yogurt.

Lee's HiVeg Agar

Agar 18.0g

Yeast extract 10.0g

Lactose 5.0g

Sucrose 5.0g

CaCO3 3.0g

K2HPO4 0.5g

pH 7.0 ± 0.2 at 25°C

HiMe-dia

Gen-tly heat and bring to boiling Autoclave for 20 min at 15 psi pressure–

121°C Mix thoroughly Pour into sterile, chilled Petri dishes in 20–

25mL volumes Swirl flask while dispensing to evenly suspend CaCO3 Dry plates at 30°C for 18–24 hr before use

Use: For the isolation, cultivation, and enumeration of Lactobacillus bulgaricus from yogurt.

Lee's Multidifferential Agar

Tomato juice, dessicated 20.0g Peptonized milk 20.0g Glucose 10.0g Yeast extract 10.0g Agar 15.0g CaCO3 5.0g Calcium pantothenate 2.0g Citric acid 1.1g Polysorbate 80 0.5g

K2HPO4 0.5g

KH2PO4 0.5g MgSO4·7H2O 0.2g FeSO4·7H2O 0.01g MnSO4·7H2O 0.01g NaCl 0.01g Bromcresol Green 0.022g Cycloheximide 7.0mg

pH 5.5 ± 0.2 at 25°C

Caution: Cycloheximide is toxic Avoid skin contact or aerosol for-mation and inhalation

or flasks Autoclave for 10 min at 15 psi pressure–121°C Pour into sterile Petri plates while swirling to prevent calcium carbonate from settling The medium will have a white precipitate of calcium carbon-ate

Use: For the detection of most organisms commonly encountered in a brewery Acid producing bacteria are identified by the development of

a clear zone around the colonies Further identification is facilitated by the characteristic color reactions

Lee's Multidifferential HiVeg Agar

Tomato juice, dessicated 20.0g Plant hydrolysate No 3 20.0g Agar 15.0g Glucose 10.0g Yeast extract 10.0g CaCO3 5.0g Calcium pantothenate 2.0g Citric acid 1.1g Polysorbate 80 0.5g

K2HPO4 0.5g

KH2PO4 0.5g MgSO4·7H2O 0.2g FeSO4·7H2O 0.01g MnSO4·7H2O 0.01g NaCl 0.01g Bromcresol Green 0.022g Cycloheximide 7.0mg

pH 6.7 ± 0.2 at 25°C

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942 Legionella Agar Base

HiMe-dia

Caution: Cycloheximide is toxic Avoid skin contact or aerosol

for-mation and inhalation

or flasks Pour into sterile Petri plates while swirling to prevent calcium

carbonate from settling The medium will have a white precipitate of

calcium carbonate

Use: For the detection of most organisms commonly encountered in a

brewery Acid producing bacteria are identified by the development of

a clear zone around the colonies Further identification is facilitated by

the characteristic color reactions

Legionella Agar Base (Legionella Medium)

Agar 17.0g

Yeast extract 10.0g

ACES buffer

(N-2-acetamido-2-aminoethane sulfonic acid) 6.0g

Charcoal, activated 1.5g

KOH 1.5g

α-Ketoglutarate 1.0g

Legionella agar enrichment 10.0mL

pH 6.85–7.0 at 25°C

Source: This medium is available as a prepared medium from BD

Di-agnostic Systems

Legionella Agar Enrichment:

Fe4(P2O7)3 0.25g

Preparation of Legionella Agar Enrichment: Add components

to distilled/deionized water and bring volume to 10.0mL Mix

thor-oughly Filter sterilize

Preparation of Medium: Add components, except Legionella agar

enrichment, to distilled/deionized water and bring volume to 990.0mL

Mix thoroughly Gently heat to boiling Autoclave for 15 min at 15 psi

pressure–121°C Cool to 50° C Add 10.0mL of sterile Legionella agar

enrichment Adjust pH to 6.9 at 50°C by adding 4.0–4.5mL of 1.0N

KOH This is a critical step Mix thoroughly Pour into sterile Petri dishes

in 20.0mL volumes Swirl medium while pouring to keep charcoal in

suspension

Use: For the preparation of Legionella agars For the isolation and

cul-tivation of Legionella species from clinical and nonclinical materials.

Legionella pneumophila Medium

(Charcoal Yeast Extract Diphasic

Blood Culture Medium) (Diphasic Blood Culture Buffered

Charcoal Yeast Extract Medium)

(CYE-DBCM)

Agar phase 500.0mL

Broth phase 500.0mL

pH 6.9–7.0 at 25°C

Agar Phase:

Agar 17.0g Charcoal, activated 2.0g

Preparation of Agar Phase: Add components to distilled/deion-ized water and bring volume to 500.0mL Mix thoroughly Gently heat and bring to boiling Distribute in 20.0mL volumes into 125.0mL se-rum bottles with aluminum crimp seals and rubber stoppers Autoclave for 20 min at 15 psi pressure–121°C Cool to 50°C Swirl medium to put charcoal in suspension Allow agar to solidify so that a slant with a 6.0cm height is formed

Broth Phase:

Yeast extract 20.0g L-Cysteine·HCl·H2O solution 0.4g Fe(NO3)3·9H2O solution 0.1g

Preparation of Broth Phase: Add yeast extract to distilled/deion-ized water and bring volume to 480.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to 25°C Aseptically add sterile L-cysteine·HCl·H2O solution and Fe(NO3)3·9H2O solution Mix

thoroughly Adjust pH to 6.9 with 6.0mL of sterile 1N KOH

L- Cysteine·HCl·H 2 O Solution:

L -Cysteine·HCl·H2O 0.04g

Preparation of L- Cysteine·HCl·H 2 O Solution: Add L-cyste-ine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL Mix thoroughly Filter sterilize

Fe(NO 3 ) 3 ·9H 2 O Solution:

Fe(NO3)3·9H2O 0.04g

Preparation of Fe(NO 3 ) 3 ·9H 2 O Solution: Add Fe(NO3)3·9H2O

to distilled/deionized water and bring volume to 10.0mL Mix thor-oughly Filter sterilize

Preparation of Medium: Add 20.0mL of sterile broth phase to 125.0mL serum bottles containing 20.0mL of solidified agar phase Seal bottles by crimping metal caps over rubber stoppers

Use: For the isolation and cultivation of Legionella pneumophila from

blood cultures

Legionella Selective Agar

Agar 15.0g ACES (2-[(2-amino-2-oxoethyl)-amino]ethane

sulfonic acid) buffer 10.0g Yeast extract 10.0g Charcoal, activated 2.0g α-Ketoglutarate 1.0g L-Cysteine·HCl·H2O solution 10.0mL

Fe4(P2O7)3 solution 10.0mL Antibiotic solution 10.0mL

pH 6.85–7.0 at 25°C

Source: This medium is available as a prepared medium from BD Di-agnostic Systems

L -Cysteine·HCl·H 2 O Solution:

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Leishmania Medium 943

Preparation of L -Cysteine·HCl·H 2 O Solution: Add

L-cyste-ine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL

Mix thoroughly Filter sterilize

Fe 4 (P 2 O 7 ) 3 Solution:

Fe4(P2O7)3 0.25g

Preparation of Fe 4 (P 2 O 7 ) 3 Solution: Add Fe4(P2O7)3 to distilled/

sterilize

Anisomycin 10.0mg

Colistin 3.75mg

Vancomycin 2.0mg

sterilize

Preparation of Medium: Add components—except

L-cyste-ine·HCl·H2O, Fe4(P2O7)3, and antibiotic solutions—to distilled/deionized

water and bring volume to 970.0mL Mix thoroughly Gently heat and

bring to boiling Autoclave for 15 min at 15 psi pressure–121°C Cool to

45°–50°C Aseptically add sterile L-cysteine·HCl·H2O, Fe4(P2O7)3,and

antibiotic solutions Mix thoroughly Pour into sterile Petri dishes Swirl

medium while pouring to keep charcoal in suspension

Use: Legionella selective agar is used in qualitative procedures for the

isolation of Legionella species from clinical and nonclinical

speci-mens

Legume Extract Agar

Alfalfa roots 35.0g

Agar 20.0g

Soybean meal 10.0g

Sucrose 10.0g

CaCO3 5.0g

Glucose 5.0g

K2HPO4 1.0g

MgSO4·7H2O 0.2g

CaCl2 0.1g

NaCl 0.1g

FeCl3 1.0mg

Preparation of Medium: Wash the alfalfa roots well and cut them

up Add 10.0g of soybean meal Add three times the volume of

dis-tilled/deionized water Steam for 1 hr Let stand at 25°C overnight

Bring volume to 1.0L with distilled/deionized water Filter through

pa-per pulp To the filtrate, add the K2HPO4, CaCl2, MgSO4·7H2O, NaCl,

FeCl3, and agar Autoclave for 20 min at 15 psi pressure–121°C Cool

to 45°–50°C Add the CaCO3, sucrose, and glucose Mix thoroughly

Distribute into tubes or flasks Autoclave for 20 min at 10 psi pressure–

115°C

Use: For the cultivation of Rhizobium species.

Leifson HiVeg Agar

Agar 12.0g

Lactose 10.0g

Plant extract No 1 6.5g

Na2S2O3 5.4g

Plant peptone No 1 5.0g

Synthetic detergent No III 3.0g Ferric citrate 1.0g Neutral Red 0.02g

pH 7.5 ± 0.2 at 25°C

or flasks Gently bring to boiling Do not autoclave Pour into sterile Petri plates

Use: For the isolation of Salmonella and Shigella species.

Leifson Medium

Agar 15.0g Pancreatic digest of casein 2.0g MgCl2 1.0g Yeast extract 1.0g

pH 8.0 ± 0.2 at 25°C

to boiling Adjust pH to 8.0 Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C Pour into sterile Petri dishes or leave in tubes

Use: For the direct isolation and routine culturing of Hyphomonas

spe-cies

Leifson's Deoxycholate HiVeg Agar, Modified (Hugh Leifson Deoxycholate HiVeg Agar, Modified)

Agar 15.0g Lactose 10.0g Plant extract 5.0g Plant peptone 5.0g Sodium citrate 5.0g

Na2S2O3 5.0g Synthetic detergent No III 2.5g Ferric citrate 1.0g Neutral Red 0.025g

pH 7.0 ± 0.2 at 25°C

or flasks Gently bring to boiling Do not autoclave Pour into sterile Petri plates

Use: For the selective isolation and differentiation of Salmonella and Shigella species.

Leishmania Medium

Sodium citrate 1.2g NaCl 1.0g Rabbit blood solution 10.0mL

Rabbit Blood Solution:

Rabbit blood, defibrinated 5.0mL

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944 Leonian’s Agar

Preparation of Rabbit Blood Solution: Add 5.0mL of whole

rabbit blood to 5.0mL of sterile distilled/deionized water Freeze and

thaw twice to lyse blood cells

Preparation of Medium: Add components, except rabbit blood

so-lution, to distilled/deionized water and bring volume to 90.0mL Mix

thoroughly Autoclave for 15 min at 15 psi pressure–121°C

Aseptical-ly add 10.0mL of sterile rabbit blood solution Mix thoroughAseptical-ly

Asep-tically distribute into sterile, screw-capped tubes or flasks

Use: For the cultivation of Leishmania donovani, Leishmania hertigi,

and Leishmania tropica.

LEMB Agar

See: Levine EMB Agar Lenox Broth

See: LB Medium

Leonian’s Agar

Agar 20.0g

Maltose 6.25g

Malt extract 6.25g

KH2PO4 1.25g

Peptone 0.625g

MgSO4·7H2O 0.625g

water and bring volume to 1.0L Mix thoroughly Gently heat and bring

to boiling Distribute into tubes or flasks Autoclave for 15 min at 15

psi pressure–121°C Pour into sterile Petri dishes or leave in tubes

Use: For the cultivation and maintenance of Amorphotheca resinae,

Apio-sordaria rotula, Arachnotheca glomerata, Ascotricha erinacea,

Auxar-thron pseudauxarAuxar-thron, Coniochaeta extramundana, Coniochaetidium

ostreum, Coprinus velox, Cylindrocladium couratariae, Dicranidion

frag-ile, Dicranidion gracilis, Eupenicillium brefeldianum, Isaria sulfurea,

Linderina macrospora, Microthecium retisporum, Nigrospora sacchari,

Orbicula parietina, Pectinotrichum llanense, Penicillium ochrochloron,

Penicillium pinophilum, Pseudogymnoascus roseus, Thielavia terricola,

Triangularia batistae, Tripospermum myrti, and Zopfiella pleuropora.

Leptospira HiVeg Medium Base, Korthof, Modified

with Rabbit Serum

NaCl 1.4g

Na2HPO4 0.88g

Plant peptone 0.8g

KH2PO4 0.24g

CaCl2 0.04g

KCl 0.04g

NaHCO3 0.02g

Rabbit serum, heat inactivated at 56°C 100.0mL

pH 7.2 ± 0.2 at 25°C

Source: This medium, without rabbit serum, is available as a

pre-mixed powder from HiMedia

Preparation of Medium: Add components, except rabbit serum, to

distilled/deionized water and bring volume to 900.0mL Mix thoroughly

Gently heat and bring to boiling Distribute into tubes or flasks

Auto-clave for 15 min at 10 psi pressure–115°C Cool to 50°–56°C

Aseptical-ly add 100.0mL of rabbit serum Mix thoroughAseptical-ly

Use: For the cultivation of Leptospira species

Leptospira HiVeg Medium Base, Korthof, Modified

with Rabbit Serum and Hemoglobin

NaCl 1.4g

Na2HPO4 0.88g Plant peptone 0.8g

KH2PO4 0.24g CaCl2 0.04g KCl 0.04g NaHCO3 0.02g Rabbit serum, heat inactivated at 56°C 80.0mL Hemoglobin solution 8.0mL

pH 7.2 ± 0.2 at 25°C

Source: This medium, without rabbit serum, is available as a pre-mixed powder from HiMedia

Hemoglobin Solution:

Composition per 50.0mL:

Bovine hemoglobin 1.0g

Preparation of Hemoglobin Solution: Add bovine hemoglobin to distilled/deionized water and bring volume to 500.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C

Preparation of Medium: Add components, except rabbit serum, and hemoglobin solution, to distilled/deionized water and bring volume

to 1.0L Mix thoroughly Gently heat and bring to boiling Distribute into tubes or flasks Autoclave for 15 min at 10 psi pressure–115°C Cool to 50°–56°C Aseptically add 80.0mL of rabbit serum and 8.0mL of hemo-globin solution Mix thoroughly Aseptically dispense into tubes

Use: For the cultivation of Leptospira species

Leptospira Medium

(NH4)2Fe(SO4)2·6H2O 6.0g NaH2PO4 0.53g L-Asparagine 0.5g Glycerol 0.2g Tween™ 60 0.2g MgSO4·7H2O 0.15g

KH2PO4 0.069g Tween™ 80 0.05g EDTA 0.01g CaCO3 4.0mg Thiamine·HCl 1.0mg Vitamin B12 1.0μg

pH 7.4–7.6 at 25°C

Preparation of Medium: Add components, except thiamine·HCl,

to distilled/deionized water and bring volume to 990.0mL Mix thor-oughly Gently heat and bring to boiling Autoclave for 15 min at 15 psi pressure–121°C Aseptically add 1.0mg of thiamine·HCl Aseptically distribute into sterile tubes or flasks

Use: For the cultivation of Leptospira species.

Leptospira Medium

(DSMZ Medium 1113)

Agarose 1.5g

Na2HPO4 1.0g

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